PROJECT SUMMARY Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world?s population and kills over a million people annually. Although it is known that CD4 T cells and interferon (IFN)? are crucial for defense against Mtb, a general lack of understanding of how this response is regulated has thus far hampered successful vaccine development. Our lab focuses on the CD4 T cell response to two immunodominant Mtb antigens: ESAT6, which is expressed continuously, and Ag85B, which is expressed only during bacterial replication. We have found that differential exposure of these T cells to their cognate antigens in the lung results in functional exhaustion of ESAT6-specific T cells but not Ag85B-specific T cells. An unbiased bioinformatics approach predicted vitamin D receptor (VDR) as a regulator of a subset of genes that are enriched in Ag85B-specific T cells, leading us to investigate the role of T cell VDR signaling during Mtb infection. Indeed, VDR deficiency had differential effects on Mtb-specific CD4 T cells in an in vivo mouse infection model: ESAT6-specific T cells failed to expand, yet Ag85B-specific T cells had enhanced IFN? production. These results have interesting implications for Mtb therapeutics because low vitamin D levels have long been associated with increased susceptibility to disease, although the role of vitamin D signaling in T cells has never been explored in the context of tuberculosis. The central hypothesis of this proposal is that VDR signaling in both T cells and macrophages plays an essential role in protection from tuberculosis. This hypothesis will be tested using a combination of systems biology and cellular immunology approaches. More specifically, Aim 1 will seek to identify VDR target genes of ESAT6- and Ag85B-specific T cells by comparison of WT and VDR-/- T cell transcriptomes using RNA-seq. Aim 2 will address the question of how VDR is controlling the maintenance of Mtb-specific T cells and whether it is indeed dependent on antigen availability. Finally, Aim 3 will use T cell-specific VDR knockout mice and mixed bone marrow chimeras to simultaneously assess the contributions of myeloid cells and T cells to vitamin D-mediated protection. Combined, the studies proposed here will reveal the molecular networks targeted by vitamin D and elucidate how T cell VDR signaling influences the outcome of Mtb infection.